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Here at last, is
a synopsis of the responses which I received from the readers of this
esteemed list, to my recent enquiry concerning suggestions for potentially
suitable fluorescent dyes for the tracking of labelled mouse tumour
cells by flow. Firstly, let me thank all of you who took the trouble to
send replies, which is what makes this list such a potent medium for the
valuable exchange of information & ideas.
So, on to the responses received:-
(i) PKH26-GL (red-orange flourescent) & PKH2-GL (Green floursescent)
Linker dyes (Sigma): ten votes.
This intercalates into the phospholipid cell membrane, probably is
stable for the life of the cell & divides with it during mitosis (halving the
subsequent fluorescence).
Comments included:-
Does not leak.
Can be used together with surface marker antibodies.
May be toxic (eg. to sheep lymphoid cells).
Labelling conditions may vary & must be optimised for experimental
requirements & cell lines.
Needs careful titration to avoid potential serious disturbance to cell
membranes.
Has been used successfully to follow lymphocytes infiltrating spinal
columns in mice for 7 - 10 days after injection.
Available only as kits, not particularly inexpensive.
(ii) Carboxyflourescein diacetate (Molecular Probes): three votes.
Membrane-permeant (dicetate) until intracellular esterases convert it
into carboxyfluorescein which is charged & leaks out only slowly. Carboxy-FDA
less pH-sensitive than plain flourescein.
Comments:-
Very bright, so much so that other probes may not be measurable also.
(iii) Carboxyfluorescein succinimidyl ester (M. Probes): four votes.
Attaches covalently to intracellular proteins. Binds inside the cell
via. the succinimide.
Comments:-
Has been used to enumerate proliferative generations.
Reference search: try Parish, C.
(iv) Biotin/Biotinylation: three votes.
Comments:-
Long-lived in RBCs (many weeks after re-injection in vivo).
Detection with streptavidin-FITC gives good signal/noise ratio (eg.
1/1,000 easily with 3% precision).
Harmless to cells.
Permits both cell detection & recovery, if needed.
Has been used to follow RBCs in monkeys, in the 0.05% range.
See: Ault, K.A. & Knowles, C. - Exp. Haematology. 23:996-1001. 1995.
Also: "Red cells & reticulocytes": Ch. 8, "Flow cytometry: Clinical
Applications". Ed. M. Macey. Blackwell.
(v) Calcein-AM: one vote.
Comments:-
Loads in 15min.
One of the brightest dyes used for cell-tracking.
Observed loss: <5% in 4hrs.
(vi) 5,6 - Carboxy dimethyl fluorescein diacetate (Sigma): one vote.
Comments:-
Simple to work with.
Stains viable cells for a long time.
Will not photobleach significantly.
(vii) Green BODIPY dye (M. Probes): one vote.
(viii) BCECF: one vote.
Well, that is about it. Hopefully this may be of use to some of you...
Once again, thanks to all concerned.
May your lasers never dim & your orifices never clog!
Regards,
"Zip".
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